An optimized version of the Secretome protein enrichment with click sugars (SPECS) method leads to enhanced coverage of the secretome

ABSTRACT: The secretome, the entirety of all soluble proteins either being secreted or proteolytically released by a cell, plays a key role in intercellular communication of multicellular organisms. Pathological alterations contribute to diseases such as hypertension, cancer, autoimmune disorders or neurodegenerative diseases. Hence, studying disease related perturbations of the secretome and the secretome itself covers an important aspect of cellular physiology. We recently developed the Secretome Protein Enrichment with Click Sugars (SPECS) method which enables the analysis of secretomes of in vitro cell cultures even in the presence of fetal calf serum with mass spectrometry and thus far enabled the identification of protease substrates of BACE1, SPPL3 and ADAM10. Though, the SPECS method has already enabled deep insights into secretome biology, we aimed to improve the SPECS protocol to obtain even more information from mass spectrometry based secretome analysis and reduce the amount of input material. Here, we optimized pH and the reaction buffer and replaced DBCO-PEG12-Biotin with the more water soluble variant DBCO-Sulfo-Biotin to finally provide an optimized protocol of the recently published SPECS protocol which increases the number of quantified glycoproteins 1.6-fold and their sequence coverage 2.4-fold and thus allows to reduce the input material by half without losing information. These improvements make the SPECS method more sensitive and more universal applicable to low abundant cell types.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Kidney Cell

SUBMITTER: Stephan Mueller  

LAB HEAD: Peer-Hendrik Kuhn

PROVIDER: PXD004555 | Pride | 2017-11-24

REPOSITORIES: Pride